Atrial fibrillation (AF) is the most prevalent type of cardiac arrhythmia in the developed world, affecting 6 million people in the United States alone, a number that is expected to double by 2020 because of population aging. Current pharmacological options are ineffective, which may be in part because these drugs fail to target key pathogenic mechanisms. Recent work has shown that dysfunction of the cardiac ryanodine receptor (RyR2) plays a central role in AF pathogenesis. Diastolic leak of calcium (Ca2+) via RyR2 can initiate triggered activity, which can initiate new AF episodes, and can also promote atrial remodeling underlying the perpetuation of AF. Our long-term goal is to develop potent anti-arrhythmic drugs to treat AF and other arrhythmias. The objective is to obtain pre-clinical data showing that Elex Biotech's promising new lead compounds embody effective pharmaceuticals for the treatment of AF. Our central hypothesis is that drugs with enhanced electron donor properties that target RyR2 will be highly effective in decreasing the SR Ca2+ leak associated with atrial arrhythmias. Building on our recent work demonstrating anti-arrhythmic properties in animal models of ventricular tachycardia, we will test our compounds in clinically relevant animal models of AF. In Specific Aim 1, we will optimize lead compounds using an SAR evaluation based on in vitro assays. In Specific Aim 2, we will perform a preclinical evaluation of lead drug candidates in a mouse model of progressive AF. In Specific Aim 3, we will characterize the effects of novel RyR2 inhibitors on AF in tachypaced rabbits. Significance: These studies are expected to identify outstanding leads for further pre-clinical studies (e.g., large animal models of AF) and eventual clinical development. Compound selection will be based on potency defined as equal or better than and IC50 <100 nM for inhibition of Ca2+ spark frequency in atrial myocytes, and in vivo efficacy at <10 g/kg or less in a mouse model of AF.